In hippocampal CA1 pyramidal neurons, small conductance Ca2+-activated K+ channels (SK channels), type 2, SK2 channels, are expressed in dendrites and in the post-synaptic membrane of dendritic spines where they are activated by synaptically evoked Ca2+ influx. Their repolarizing influence shapes excitatory postsynaptic potentials (EPSPs), spine Ca2+ transients, and modulates the induction of synaptic plasticity. Moreover, activity that induces long-term potentiation (LTP) causes PKA-dependent SK2 channel endocytosis, a process that requires prior exocytosis of GluA1-containing AMPA-type glutamate receptors, thereby contributing to the expression of LTP. These cellular roles are reflected by behaviors, as SK2 channel activity modulates learning. The SK2 gene encodes two isoforms, SK2-L and SK2-S, and in mice that selectively lack SK2-L, synaptic localization and function are abolished. We have recently found that two synaptic scaffold proteins, MPP2 and SAP97, specifically co-assemble with SK2 channels as well as GluA1-containing AMPARs in brain and when they are heterologously co-expressed. Importantly, knocking down MPP2 expression abolished synaptic SK2 channel function. Taken together, these results engender a working model and our overriding hypothesis: MPP2 and SAP97 are essential for the physiological roles of synaptic SK2-containing channels, including synaptic transmission, plasticity, and co-ordinate LTP-dependent SK channel-AMPAR trafficking. We will use an innovative strategy together with an integrated technical repertoire to address the following specific hypotheses. 1. MPP2 and SAP97 co-assemble with SK2 before but not after LTP induction. A) Determine whether the interactions between SK2 and MPP2 or SAP97 are LTP-dependent. B) Determine the sub-spine spatial relationships between SK2, GluA1, MPP2, and SAP97 before and after LTP. 2. i) Co-assembly of SK2 and MPP2 or SAP97 is mediated by SH3 interactions, and occluded by PKA, ii) PDZ interactions mediate co-assembly of GluA1 and MPP2 or SAP97, iii) L27 interactions mediate co-assembly of MPP2 and SAP97. A) Map the interactions between SK2 or GluA1 and MPP2 or SAP97. B) Map the interactions between MPP2 and SAP97. C) Determine whether PKA phosphorylation of SK2 abrogates the interaction with MPP2 or SAP97. 3. MPP2 and SAP97 are necessary for synaptic SK channel and AMPAR function, trafficking and LTP. A) Use shRNA expression in CA1 pyramidal neurons to knockdown MPP2 or SAP97 and re-express shRNA-immune wild type or interaction-disabled MPP2 or SAP97, B) Load cells with competitor peptides to acutely and selectively block domain-specific interactions. Assess: i) the SK channel contribution to EPSPs, before and after LTP, ii) the magnitude of LTP, iii) the AMPA:NMDA ratio. 4. Dendritic SK2 channels influence synaptic integration. Determine the effects of SK2 channel activity on A) E-S coupling and B) summation. These proposed studies will elucidate the molecular mechanisms that engender proper and dynamic localization of SK2-containing channels, essential for normal neurotransmission, plasticity, and learning.